Lecture 11
Eukaryotic gene regulation:
Co-transcriptional and post-transcriptional
modifications of pre messenger RNA - I
mRNA synthesis and processing
5’
3’
AAAAAAAAAA
mRNA capping
mRNA splicing
-------------------
Polyadenylation
Mr. pre-mRNA,
first, put your cap on,
get spliced up,
then wag your tail
EXON
EXON
EXON
EXON
EXON
EXON
A
A
A
A
A
A
A
A
A
SEVERAL POST TRANSCRIPTIONAL MODIFICATIONS OF PRE mRNA ARE
CO-TRANSCRIPTIONAL , RATHER THAN POST-TRANSCRIPTIONAL
In the nucleus, mRNA capping, splicing and polyadenylation occur at the
same time and place, in close proximity
The target for factors involved in RNA processing is not a solitary RNA molecule
Instead, the transcription elongation complex (TEC) comprising the growing
nascent RNA, RNA polymerase and proteins associated with it are targeted by a
plethora of factors involved in RNA processing.
RNA Polymerase II C-terminal domain (CTD) plays a key
role in pre-mRNA processing.
Hypophosphorylated CTD (pol IIA)
Hyperphosphorylated CTD (pol IIO)
Mutations in CTD affect not only transcription initiation but
also certain steps of RNA processing
RNA Polymerase II CTD acts as a ‘‘landing pad,’’ for the
binding of factors involved in pre-mRNA capping, 3’ end
processing, transcription elongation, termination, and
chromatin modification
TRANSCRIPTION ELONGATION COMPLEX
(TEC)
HOW DOES TRANSCRIPTION ELONGATION OCCUR?
HOW IS IT REGULATED?
HOW ARE TRANSCRIPTION ELONGATION, CAPPING,
SPLICING AND POLYADENYLATION ARE INTEGRATED?
RNA Pol II CTD contains heptad repeats (26 in yeast, 52 in mammals)
with the consensus sequence :
*
*
*
**
*
The RNA polymerase II CTD interacts directly with a number of proteins involved in
mRNA processing :
i)Cgt1, an enzyme involved in mRNA capping
ii)Pcf11, a protein involved in polyadenylation
iii)Set2, a histone methyltransferase and
iv)Nrd1, an RNA binding factor involved in transcription termination
The heptad repeats are phosphorylated and dephosphorylated by
kinases and phosphatases at the S2, S5, and S7 positions during the
various phases of initiation, elongation, and termination cycles.
*
*
*
*
*
*
Phosphorylated RNA Pol II CTD was shown to allosterically activate the
guanylyl transferase activity of mRNA capping enzyme.
At the time of transcription initiation, S5 is phosphorylated by the TFIIHassociated kinase Cdk7 (Kin 28 in yeast).
During transcription elongation, S2 is phosphorylated by Cdk9/PTEFb, a
factor involved in transcription elongation
The S5 and S2 residues are dephosphorylated by the Rtr1 and Fcp1
phosphatases during different stages of mRNA synthesis and processing.
The S7 residues of the CTD heptads are also phosphorylated by
Kin28/Cdk7 in yeast and mammalian cells and this results in the
recruitment of factors Involved in transcription elongation such as Nrd1.
Pre-mRNAs are modified at their 5’ ends by addition of a 7-methyl G5’ppp5’N cap when
the pre-mRNA is only 25–50 bases long.
The 5' cap is a unique feature of eukaryotic cellular and viral
messenger RNAs that is absent in prokaryotes
The cap structure
• discovery: A. Shatkin in 1976
• found on all cellular cytoplasmic mRNAs.
• Mitochondrial mRNAs and a few viral RNAs do not have it
Cap structure: the first base (B1) is usually a purine.
Capping is a three-step process
• Removal of the g-phosphate of the first
nucleotide by RNA triphosphatase.
• Addition
of
GMP
by
guanylyltransferase in two steps:
ppRNA + Pi
RNA
i) a lysine side chain on the enzyme (E)
reacts with the phosphorus of GTP to form
a covalent enzyme–guanylate intermediate
(EpG) . Pyrophosphate (PPi) is released.
ii) enzymatic transfer of GMP to the 5'
diphosphate RNA end to form a G cap and
regeneration of the apoenzyme.
• Methylation of guanine at N7
position by guanine N7
methyltransferase.
pppRNA
E + pppG
EpG + PPi
E + ppRNA
GpppRNA + E
GpppRNA + AdoMet
m7GpppRNA
+ AdoHcy
E + pppG
EpG + PPi
E + ppRNA
GpppRNA + E
The guanylyltransferase acts specifically on 5' diphosphate RNA ends, and it does
not catalyse the transfer of GMP to a 5' monophosphate RNA.
Thus, caps are added only to the 5' end of the pre-mRNAs and not to processed 5'
ends that arise from endonucleolytic cleavage of mRNAs.
Prokaryotic 'mRNAs contain an eight-nucleotide Shine–Dalgarno sequence,
which is located proximal to the site of translation initiation.
This sequence can base-pair with the sequence near the 3' end of 16S rRNA of
the bacterial ribosome, and position the AUG start codon at the correct site on
the ribosome.
Eukaryotes use the cap as an alternative signal to direct the translation
apparatus to the 5' end of protein-encoding RNAs.
A protein known as the m7G cap-binding protein (eIF4E) binds to the 5’ cap and
recruits the 40S ribosome subunit to the 5' end of the mRNA.
Role of RNA polymerase II CTD in mRNA capping
Guanylyltransferase and methyltransferase do not associate with one another
How are these two enzymes brought to the vicinity of the RNA substrate?
Both these enzymes bind directly and specifically to the phosphorylated Pol II CTD.
When transcription initiates, phosphorylation of the CTD on S5 residue permits
loading of capping enzyme onto the TEC and allosteric activation of the
guanylyltransferase.
Thus, the capping reaction is facilitated by both colocalization of the capping
enzymes on the phosphorylated CTD and allosteric activation.
Is mRNA capping a regulatory step in transcription?
Capping enzymes stimulate or inhibit transcription initiation and early elongation
There is a phenomenon called 5’ pausing wherein, transcription pauses after initiation.
5’ pausing is regulated by proteins such as transcription elongation factor Spt5 and HIV1
Tat protein which activate the guanylytransferase and enhance capping and promote
transcription elongation.
Thus, CTD phosphorylation and recruitment of factors such as Spt5 act as key regulatory
steps in transcription elongation and mRNA capping.
The capping enzymes continue to be associated with the RNA polymerase II even after
capping and thus may have a regulatory role in other RNA processing activities as well.
THUS, FACTORS INVOLVED IN TRANSCRIPTION
ELONGATION AND mRNA PROCESSING ALSO PLAY
A ROLE IN REGULATION OF mRNA SYNTHESIS
Interrelationship between mRNA capping enzyme, RNA polymerase CTD and
transcription elongation
Spt5
(elongation
factor)
*
*
S2, S5 phosphorylation of CTD
CE
Guanylation of pre mRNA
Shuman, S. (2001). Structure, mechanism, and evolution of the mRNA capping
apparatus. Prog. Nucleic Acid Res. Mol. Biol. 66, 1–40.
Shuman, S. (2002) What messenger RNA capping tells us about eukaryotic evolution.
Nat Rev Mol Cell Biol. 3, 619-25.
Interactions between RNA Polymerase
and
mRNA splicing machinery
The multi ribonucleoprotein complex involved in mRNA splicing is called
spliceosome. There are five snRNAs in a spliceosome (U1 to U6) and as
many as 100 proteins.
5’ splice site (donor)
branch site
3’ splice site (acceptor)
5’ ….. AGGURAGU…………….A…………….NNNNNNNNAGGUCC…. 3’
5’ …C/A AG GU A/G AGU……………..A………….
CONSENSUS SEQUENCE
FOR 5’ SPLICE SITE
BRANCH
POINT
UUUUUUUUUUU
CCCCCCCCCCC
CONSENSUS SEQUENCE FOR
3’ SPLICE SITE
GURAGU…………….A…………….NNNNNNNNAG
5’ …..AGGUCC 3’…..
N C/U AG A/G…3’
INTRON
SPLICED EXONS
mRNA splicing
Cotranscriptional or post transcriptional ?
Beyer AL, Osheim YN.
Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.
Genes Dev. 1988 Jun;2(6):754-65.
These authors demonstrated for the first time by electron microscopy that many,
but not all, introns are removed cotranscriptionally rather than posttranscriptionally.
Interactions between mRNA splicing machinery and RNA Pol II
Yeast U1 snRNP protein Prp40, which bridges the 5’ splice site and branch
point, was shown to bind directly to the phosphorylated CTD.
Human U1snRNP, but not other snRNPs, also coimmunoprecipitates with
RNA Pol II.
U1snRNP at a 5’ splice site can promote recruitment of RNA Pol II and
general transcription factors to the promoter indicating that there is a special
relationship between transcription initiation/elongation machinery and
components of mRNA splicing.
Proteins known as SR (serine-arginine rich) proteins involved in mRNA
splicing were shown to interact with RNA Pol II and promote transcription
elongation.
Several elongation factors such as PTEFb, CA150, and TAT-SF1 associate
directly or indirectly with spliceosomal U snRNPs
A functional role for RNA Pol II CTD in splicing
Transcriptional activation of pol II genes induces the association of splicing
factors to sites of transcription only when RNA pol II has a full CTD
Deletion of the RNA Pol II CTD inhibits splicing of the β-globin gene
Using in vitro splicing reactions it was demonstrated that phosphorylation
of RNA polymerase II CTD enhances mRNA splicing.
Two examples:
1.Role of RNA pol II CTD in splicing
2.Role of transcription elongation in splicing
Role of RNA pol II CTD in generating two different forms of mRNA
by alternate splicing in case of fibronectin gene
weak 3’ SS
strong 3’ SS
X
Exon I
Exon II
Exon III
RNA Pol
II
SRp20
Exon I
Exon III
weak 3’ SS
Exon I
Exon skipping
strong 3’ SS
Exon II
Exon III
RNA Pol
II
X
SRp20
Exon I
Exon II
Exon III
Exon inclusion
The rate of transcription elongation in eukaryotes varies from 1.9 kb/min to 4.3 kb/min
The rate of elongation may often influence splicing and can lead to alternate splicing.
weak 3’ SS
Exon I
strong 3’ SS
Exon II
Exon III
Fast elongation
Exon I
Slow elongation
Exon III
Exon skipping
Exon I
Exon II
Exon inclusion
Exon III
Misteli, T., J.F. Caceres, and D.L. Spector. 1997.
The dynamics of a pre-mRNA splicing factor in living cells.
Nature 387: 523-527
Following activation of a reporter gene in cells expressing either full-length or CTD-truncated
RNAP II as the only source of active enzyme, sites of accumulation of both the newly
synthesized reporter transcripts and splicing factors were simultaneously visualized by
immunohistochemistry techniques.
Although both sites colocalized well in cells expressing wild-type RNAP II, the transcription
sites did not colocalize with either SR proteins or snRNP particles in cells expressing the
CTD-truncated RNAP II.
Truncation of the CTD prevented accumulation of spliced products despite the presence of
significant amounts of unspliced pre-mRNAs.
These results supported the idea that the CTD is required for targeting splicing factors to
transcription sites and that this can be important for efficient splicing.
Original research articles
In response to UV-induced DNA damage, the CTD becomes hyperphosphorylated,
transcription elongation slows down, and alternative splice choices are switched in favor of
the proapoptotic isoforms of Bcl-x and caspase.
Munoz, M.J., et al., (2009).
DNA damage regulates alternative splicing through inhibition of RNA polymerase II
elongation. Cell 137, 708–720.
S. McCracken et al., The C-terminal domain of RNA polymerase II couples mRNA
processing to transcription, Nature 385 (1997), pp. 357–361.
McCracken et al., 5′-Capping enzymes are targeted to pre-mRNA by binding to the
phosphorylated carboxy-terminal domain of RNA polymerase II, Genes Dev. 11 (1997),
pp. 3306–3318
de la Mata, M. et al., (2003).
A slow RNA polymerase II affects alternative splicing in vivo.
Mol. Cell 12, 525–532.
M. de la Mata and A.R. Kornblihtt
RNA polymerase II C-terminal domain mediates regulation of
alternative splicing by SRp20 Nat. Struct. Mol. Biol. 13 (2006), 973–980.